The initial reaction of steroidogenesis is the cleavage of the cholesterol side chain by a specific cytochrome P-450 located on the inside face of the inner mitochondrial membrane. The resultant steroid, pregnenolone, is then metabolized by enzymes having extramitochondrial locations. Thus, pregnenolone must move out of the mitochondria, crossing the organelle's inner and outer membranes. Despite the significance of pregnenolone efflux from mitochondria, the process remains poorly characterized. This process is currently being investigated with the guinea pig adrenal cortex model. In this model there exists a specific pregnenolone- binding protein (PBP). Although PBP behaves as a Mr 58,000 globular protein on gel permeation chromatography, on SDS PAGE it migrates as a Mr 34,000 protein. A polyclonal antibody has been generated against the 34 kDa protein. At all stages of purification, including the starting material, Western blot analysis of isoelectric focusing gels reveals the same pattern of apparent microheterogeneity with pIs of 6.8, 6.6, 6.4, 6.2. The major dilemma at the moment is to distinguish between microheterogeneity of a single protein and a co-purifying contaminant. Additional purification steps are under investigation, and an effort to generate an N-terminal sequence is also under way. Studies with the polyclonal antibody demonstrate that the PBP is present only in the soluble fraction of the adrenal cortex. Immunocytochemistry indicates that the PBP is most abundant in the outer adrenocortical zone (the guinea pig adrenal cortex can be divided into an ACTH-responsive outer zone and an ACTH-unresponsive inner zone). The latter finding is quite interesting for the pregnenolone-binding activity is far greater in the inner zone. It thus, appears that there are active and inactive forms of the binding protein. The latter phenomenon is also under investigation. It is possible that the active/inactive forms and the microheterogeneity are related. Success with the N- terminal sequencing will resolve the problem of purity unless the PBP is composed of two non-identical subunits (it is probable that the PBP exists as dimer in its native form). Once the polyclonal antibody specificity has been ascertained, it will be used to isolate the PBP mRNA. The ultimate goal is to develop a cDNA clone for the PBP and determine the complete amino acid PBP sequence.